The present invention relates generally to compounds, compositions and methods regulating the actions of androgens and other steroid hormones by modulating the activity of 5.alpha.-reductase. More particularly, the present invention relates to the use of these compounds to treat disorders that are caused by excess androgen action in cells or organs. 2. Description Of The Related Art
A. Steroid Hormones and Their Receptors
Androgens are one of the six major classes of steroid hormones. Steroid hormones form complexes with specific receptor proteins in selective cells of target organs [Jensen et al., Proc. Nat'l Acad. Sci.(USA), 59:632 (1968); Liao, Intl. Rev. Cytology 41:87 (1975); Gorski, et at., Ann. Rev. Physiol. 42:17 (1976)]. Steroid receptors are members of a superfamily of transcription factors that can regulate gene expression, and this function is dependent on the binding of a specific hormonal ligand to an appropriate receptor [Evans, Science 240:889 (1989); Beato, Cell 56:335 (1989); O'Malley, Mol. Endocrinol. 4:363 (1990)].
Studies of the specificity and affinity of steroid hormones for their receptors have contributed greatly to the understanding of the relationships among steroid and receptor structures and biological activity, target organ specificity, and the mechanism of action of many antihormones, including "competitive antiandrogens". "Competitive Antiandrogens" are defined herein as those antiandrogens that interact with receptors and competitively prevent receptor binding of active androgens [Fang and Liao, Mol. Pharmacol. 5:428 (1969); Liao et al., J. Biol. Chem. 248:6154 (1973); Liao et al., Endocrinology 94:1205 (1974); Chang and Liao, J. Steroid Biochem. 27:123 (1987); Liao et al., J. Steroid Biochem. 34:41 (1989)], although it should be noted that some compounds with an antiandrogenic activity may act by a different mechanism.
B. Androgen Actions
Androgen, produced in the testis, stimulates the differentiation of the male reproductive organs, including the penis, scrotum, prostate, seminal vesicles, epididymis, and vas deferens. With the onset of puberty, an increase in the production of androgen promotes the growth of these tissues. Androgen is required for spermatogenesis and accelerates skeletal muscular growth and bone formation. In the central nervous system, it stimulates libido and produces feedback inhibition of gonadotropin secretion. In skin, androgen increases the size of sebaceous glands and apocrine glands and converts villus hairs in the axillae, pubic region, and the beard to form coarser and longer terminal hairs. Androgen causes thickened vocal cords and lowers the pitch of the voice. Androgen also stimulates hematopoiesis. Uses of androgen known to the medical arts include, for example, treatment of hypogonadism and anemia [Synder, Ann. Rev. Med. 35:207 (1984); Mooradian et at., Endocrine Rev. 8:1 (1987)]. The abuse of androgen among athletes to enhance performance is well known [Strauss and Yesalis, Annu. Rev. Med. 42:499 (1991)].
Androgen is also known to promote the development of benign prostatic hyperplasia (BPH) [Wilson, Am. J. Med. 68:745 (1980)], prostate cancer [Huggins and Hodges, Cancer Res. 1:293 (1940)], baldness [Hamilton, Am. J. Anat. 71:451(1942)], acne [Pochi, Annu. Rev. Med. 41:187 (1990)], hirsutism, and seborrhea [Hammerstein et at., J. Steroid Biochem. 19:591 (1983); Moguilewsky and Bouton, J. Steroid Biochem. 31:699 (1988)]. Approximately 70% of males in the United States over the age of 50 have pathological evidence of BPH [Carter and Coffey, The Prostate 16:39-48 (1990)]. Prostate cancer is the second leading cause of cancer death in males in the United States [Silverberg and Lubera, Cancer Statistics, 40:9 (1990); Gittes, New England J. Medicine 324:236 (1991)]. Male-patterned baldness can start as early as the teens in genetically susceptible males, and it has been estimated to be present in 30% of Caucasian males at age 30, 40% of Caucasian males at age 40, and 50% of Caucasian males at age 50. Acne is the most common skin disorder treated by physicians [Pochi, Ann. Rev. Med. 41:187 (1990)] and affects at least 85% of teenagers. In women, hirsutism is one of the hallmarks of excessive androgen action [Ehrmann and Rosenfield, J. Clin. Endoerinol. Metab. 71:1 (1990)]. The ovaries and the adrenals are the major sources of androgen in women.
Differential Actions of Testosterone and 5.alpha.-Dihydrotestosterone (DHT)
In men, the major androgen circulating in the blood is testosterone. About 98% of the testosterone in blood is bound to serum proteins (high affinity binding to sex-steroid binding globulin and low affinity binding to albumin), with only 1-2% in free form [Liao and Fang, Vitamins and Hormones 27:17 (1969)]. The albumin bound testosterone, the binding of which is readily reversible, and the free form are considered to be bioavailable, and account for about 50% of total testosterone. Testosterone enters target cells, apparently by diffusion. In the prostate, seminal vesicles, skin, and some other target organs it is converted by a NADPH-dependent 5.alpha.-reductase to a more active metabolite, DHT. DHT then binds to androgen receptor (AR) in target organs [Anderson and Liao, Nature 219:277 (1968); Bruchovsky and Wilson, J. Biol. Chem. 243:2012 (1968); Liao, Int. Rev. Cytology 41:87 (1975)]. The DHT-receptor complexes interact with specific portions of the genome to regulate gene activities [Liao et al., J. Steroid Biochem. 34:41 ( 1989)]. Testosterone appears to bind to the same AR, but it has a lower affinity than DHT. In tissues such as muscle and testes, where 5.alpha.-reductase activity is low, testosterone may be the more active androgen.
The difference between testosterone and DHT activity in different androgen-responsive tissues is further suggested by findings in patients with 5.alpha.-reductase deficiency. Males with 5.alpha.-reductase deficiency are born with female-like external genitalia. When they reached puberty, their plasma levels of testosterone are normal or slightly elevated. Their muscle growth accelerates, the penis enlarges, voice deepens, and libido toward females develops. However, their prostates remain non-palpable, they have reduced body hair, and they do not develop acne or baldness. Females with 5.alpha.-reductase deficiency do not have clinical symptoms [Imperato-McGinley, Trend Genet 2:130 (1986)].
The findings in 5.alpha.-reductase deficient patients suggest that inhibitors of 5.alpha.-reductase would be useful for the treatment of prostatic cancer, BPH, acne, baldness, and female hirsutism. Clinical observations and animal experiments have indicated that spermatogenesis, maintenance of libido, sexual behavior, and feed-back inhibition of gonadotropin secretion do not require the conversion of testosterone to DHT [Brooks et al., Proc. Soc. Exp. Biol. Med. 169:67 (1982); Blohm et at., Endocrinology 119:959 (1986); George et at., Endocrinology 119:959 (1989)]. This is in contrast to other hormonal therapies which abolish the actions of both testosterone and DHT.
Treatments of androgen-dependent skin and prostatic diseases by 5.alpha.-reductase inhibitors would be expected to produce fewer side effects than the presently available hormonal therapies. These include castration, estrogen therapy, high doses of superactive gonadotropin-releasing hormone such as Luprolide, and the use of competitive antiandrogens which inhibit AR binding of testosterone and DHT, such as flutamide, cyproterone acetate and spironolactone. The long term efficacy of `competitive antiandrogens` is also compromised by their block of the androgenic feedback inhibition of gonadotropin secretion. This results in elevated gonadotropin secretion, which in turn increases testicular secretion of testosterone. The higher level of testosterone eventually overcomes the action of the antiandrogen.
D. Biological Importance of 5.alpha.-Reductase
Excessive DHT is implicated in certain androgen-dependent pathological conditions including BPH, ache, male-pattern baldness, and female idiopathic hirsutism. It has been shown that 5.alpha.-reductase activity and the DHT level are higher in the presence of BPH prostates than that of the patients with normal prostates [Isaacs, J. Clin. Endocrinol. Metab. 56:139 (1983); Siiteri and Wilson, J. Clinical Invest. 49:1737 (1970)]. 5.alpha.-Reductase activity is reported to be higher in hair follicles from the scalp of balding men than that of nonbalding men [Schweikert and wilson, Clin. Endocrinol. Metab. 38:811 (1974)].
In a given individual, 5.alpha.-reductase activity is found to be higher in balding skin than from hairy skin [Bingham and Shaw J. Endocr. 57:111 (1973)]. Some idiopathic hirsute women have a normal circulating level of testosterone, but their affected skin has a higher 5.alpha.-reductase activity than that of nonhirsute women [Serafini and Lobo, Fert Steril 43:74 (1985)]. An increased 5.alpha.-reductase activity has also been reported for skin with acne [Sansone and Reisner, J. Invest. Dermat. 56:366 (1971)].
Genetic evidence also supports the suggestion that DHT plays an important role in the development of BPH and the above skin conditions. In males with hereditary 5.alpha.-reductase deficiency, their prostates remain small or nonpalpable after puberty. They do not develop acne, temporal hairline recession, or baldness. Compared to their fathers and brothers, they have scanty beards and reduced body hair.
E. Steroidal 5.alpha.-Reductase Inhibitors
The most potent inhibitors of 5.alpha.-reductase developed so far are steroids or their derivatives. Among these the 4-azasteroidal compounds (Merck Co.) are the most extensively studied [Liang et al., J. Steroid Chem. 19:385 (1983); Rasmusson et al., J. Med. Chem. 29:2298 (1986)]. These inhibitors are 3-oxo-4-aza-5.alpha.-steroids with a bulky functional group at the 17.beta.-position, and act by reversibly competing with testosterone for the binding site on the enzyme.
The A-ring conformation of these compounds is thought to be similar to the presumed 3-enol transition state of the 5.alpha.-reduction of 3-oxo-.DELTA..sup.4 -steroids. A prototype for 5.alpha.-reductase inhibitors is 17.beta.-N,N-diethylcarbamoyl-4-methyl-4-aza-5.alpha.-androstan-3-one (4-MA), which behaves as an inhibitor of 5.alpha.-reductase in vivo, decreasing the prostatic concentration of DHT in intact male rats or in castrated male rats given testosterone propionate. 4-MA attenuated the growth of the prostate of castrated rats induced by testosterone, but had much less of an effect in rats given DHT [Brooks et al., Endocrinology 109:830 (1981)].
When dogs are treated with 4-MA, the prostate size decreases [Brooks et al., The Prostate 3:35 (1982); Wenderoth and George, Endocrinology, 113:569 (1983)]. Topical applications of 4-MA to the scalp of the stumptail macaque, a primate model of human male pattern baldness, also prevented the baldness which normally occurs at puberty in these monkeys [Rittmaster et al., J. Clin Endocrinol. Metab. 65:188 (1987)]. These results also suggest that the growth of the prostate in rats and dogs, and baldness in the stumptail macaque depend on DHT.
On the other hand, studies in rat pituitary cultures showed that complete inhibition of testosterone conversion to DHT by 4-MA did not affect testosterone inhibition of LH release, indicating direct action of testosterone in this system [Liang et al., Endocrinology 115:2311 (1984)].
Another potent inhibitor is Proscar (Merck Co.) (finasteride, MK-906, or 17.beta.-N-t-butylcarbamoyl-4-aza-5.alpha.-androst-1-en-3-one). The inhibitor has no significant affinity for the rat prostate AR. In clinical trials, Proscar decreases the plasma level of DHT and the size of the prostate and also improves urinary flow in patients with benign prostatic hyperplasia [Vermeulen et al., The Prostate 14:45 (1989); Rittmaster et al., J. Androl. 10:259 (1989); Gormley et at., J. Clin. Endocrinol. Metab. 70:1136 (1990); Imperato-McGinley et at., J. Clin. Endocrinol. Metab. 70:777 (1990)]. In stumptail macaque monkeys, Proscar administered orally at 0.5 mg/day, alone or in combination with topical 2% minoxidil, reduced serum DHT level, and reversed the balding process by enhancing hair regrowth by topical minoxidil [Diani et at., J. Clin. Endocr. and Metab. 74:345 (1992)]. The effects of Minoxidil and Proscar were additive.
Among other steroidal compounds shown to inhibit 5.alpha.-reductase are 4-androstane-3-one-17.beta.-carboxylic acid [Voigt et al., J. Biol. Chem. 260:4890 (1985)] and 4-diazo-21-hydroxymethyl-pregnane-3-one [Blohm et at., Biochem. Biophy. Res. Commun. 95:273 (1989)], and 3-carboxy A-ring aryl steroids [Brandt et at., J. Steroid Biochem. Mol. Biol. 37:575 (1990)].
F. Biological Effects of Fatty Acids and Lipids
Since treatments of androgen-dependent skin and prostatic diseases by 5.alpha.-reductase inhibitors can produce fewer side effects than the hormonal therapies which indiscriminately inhibit all androgen actions, it is desirable to provide different types of 5.alpha.-reductase inhibitors. This invention deals with the use of natural and synthetic fatty acids, especially polyunsaturated fatty acids and their derivatives as 5.alpha.-reductase inhibitors for therapeutic agents.
It is known that polyunsaturated fatty acids can correct the effects of fatty acid deficiencies that manifest as dermatitis, kidney necrosis, infertility, and cardiovascular diseases [Herold and Kinsella, Am. J. Clin. Nutr. 43:566 (1986); Phillipson et at., Eng. J. Med. 312:1210 (1985); Ziboh and Miller, Annu. Rev. Nutr. 10:433 (1990)] and also can exhibit anti-tumor activities [Begin, Proc. Nutrition Soc. 49:261 (1990); Karmali et at., J. Natl. Cancer Inst. 73:457 (1984)]. Many unsaturated fatty acids are essential components of mammalian membranes, typically in the acylated form of triglycerides and phospholipids [Lands, Ann. Rev. Biochem. 34:313 (1965)].
Arachidonic acid serves as a specific precursor in the biosynthesis of prostaglandins and leukotrienes [Needleman et at., Ann. Rev. Biochem. 55:69 (1986)]. These metabolites of unsaturated fatty acids are mediators of inflammation. Unsaturated essential fatty acids have been implicated as dietary factors that influence acne. However, no firm support for this view has developed, and no successful treatment based on this idea has appeared [Downing et at., J. Am. Acad. Dermatology 14:221 (1986)]. Synthetic retinoids and AR binding competitive antiandrogens have been used to obtain therapeutic improvement of acne in some individuals. These anti-acne agents increase the proportion of linoleic acid in sebum in parallel with clinical improvement [Wright, Prostaglandins, Leukotrienes and Essential Fatty Acids 38:229 (1989)].
G. Biochemical Effects of Fatty Acids and Lipids
Several membrane-associated enzymes (e.g., 5'-nucleotidase, acetyl CoA carboxylase) have been shown to be affected by the polyunsaturated fatty acid content of dietary fat, and to alter the physicochemical properties of cellular membranes [Zuniga et al., J. Nutr. 119:152 (1989); Szepsesi et at., J. Nutr. 119:161 (1989)]. Various types of phospholipases in rat ventricular myocytes are modulated differentially by different unsaturated fatty acids in the culture media [Nalboone et at., Lipids 25:301 (1990)]. In addition, treatment of cerebral cortical slices [Baba et at., J. Neurochem. 42:192 (1984)] or intact retina [Tesoriere et al., J. Neurochem. 51:704 (1988)] with unsaturated fatty acids can enhance adenyl cyclase activities.
Very few studies, however, have been directed to the elucidation of the mode of action of free fatty acids on enzymes in cell-free systems. Certain cis-unsaturated fatty acids, at 50 .mu.M, were shown to stimulate protein kinase C activity [Dell and Severson, Biochem. J. 258:171 (1989); Khan et at., Febs Letter 292:98 (1991)] and to inhibit steroid binding to receptors for androgens, estrogens, glucocorticoids, and progestins [Vallette et at., J. Steroid Biochem. 263:3639 (1988); Kato, J. Steroid Biochem. 34:219 (1989)]. No evidence has been presented to show that unsaturated fatty acids can affect steroid receptor binding of steroid hormones in vivo in an animal or human.
Fatty acids fluorinated at .alpha., .beta., and .omega. positions [Gershan and Parmegiani, J. Med. Chem. 10:186 (1967); Pattison and Buchanan, Biochem. J. 92:100 (1964); Gent and Ho, Biochemistry 17:3023 (1978)] and .omega.-oleic acids [Tosaki and Hearse, Basic Res. Cardiol. 83:158 (1988)] have been identified in plants and microorganisms, and have been chemically synthesized. Many of these fluorinated acids are toxic. Degradation of some fluorinated fatty acids can yield fluoro-acetic acid, which can be incorporated into fluorocitrate and can then block aconitase action. This can cause inhibition of the citric acid cycle and cellular energy production [Hail, New Phytol. 71:855 (1972)]. Fluorinated fatty acids are often useful in the studies of fatty acid degradation, metabolism and transport in biological systems [Stoll et al., J. Lipid Res. 32:843 (1991)], and biophysical studies of protein-lipid interaction and membranes functions [Gent et al., Biophys. J. 33:211 (1981)].
Biotin is a cofactor of major carboxylases which are necessary for orderly production and metabolism of fatty acids. Alopecia caused by biotin-deficiency can be completely treated by biotin administration to patients. Oral administration and cutaneous application of unsaturated fatty acids can also improve biotin-dependent dermatological conditions including scalp hair growth [Munnich et al., Lancet 2:1080 (1980); Mock et al., J. Pediatrics 106:762 (1985)]. The fatty acid effect is apparently due to supplementation of the deficient fatty acids and not related to regulation of androgen action involved in male pattern-alopecia.